Biophysics vs' Biological Physics

1
Biophysics vs. Biological Physics

• Tradition biophysics importation of
  techniques from physical sciences to study of
  biological problems frequently a one way flow
  of both ideas and people.
• Example first physicist in UC Davis Biophysics
  Grad Group in at least the last 10-15 years (in
  existence since 1961) me!

2
Biological Physics

• Ask not what physics can do for biology, but
  what biology can do for physics Hans
  Frauenfelder
• Study biological problems as interesting problems
  of the physical world.
• Practical two way flow of ideas

3
Caveat

• Just because you can throw a dog off of a roof
  to measure g does NOT mean you are doing
  biological physics L. Pelliti
• Just as physicists can study materials with
  physics ideas and methods, physicists can study
  biological systems with physics ideas and methods

4
In some ways, not new!

• 1940s Erwin Schroedinger wrote What is Life? In
  which he looked at the major problems of biology
  from the perspective of a physicist. Among other
  things-he predicted that DNA would turn out to be
  an aperiodic crystal as it did-
• The non-physicist cannot be expected to grasp -
  let alone to appreciate the
• relevance of - the difference in statistical
  structure' stated in terms so abstract
• I have just used. To give the statement life and
  colour, let me anticipate what will
• explained in much more detail later, namely, that
  the most essential part of a living
• cell - the chromosome fibre - may suitably be
  called an aperiodic crystal. In physics
• we have dealt hitherto only with periodic
  crystals. To a humble physicist's mind,
• these are very interesting and complicated
  objects they constitute one of the most
• fascinating and complex material structures by
  which inanimate nature puzzles his wits.
• Yet, compared with the aperiodic crystal, they
  are rather plain and dull. The difference
• in structure is of the same kind as that between
  an ordinary wallpaper in which the
• same pattern is repeated again and again in
  regular periodicity and a masterpiece of
• embroidery, say a Raphael tapestry, which shows
  no dull repetition, but an elaborate,
• coherent, meaningful design by the great
  master.'
• Lots of physicists were inspired at this time to
  dive into biological problems (Perutz, Crick,
  Delbruck, Monod)

5
Why an increased emphasis now?

• Biology is beginning to leave the realm of the
  qualitative and enter the realm of the
  quantitative lots and lots of data to analyze
  on the genome, on gene regulation.
• Physical science methods are providing
  fascinating new windows on biology single
  molecule studies, nanoscale microscopies..
• There is a general desire to understand
  organizing principles which physicists are pretty
  good at (why do biological proteins fold to
  compact shapes?)
• There are lots of new discoveries ALL THE TIME
  (micro rnas and their role in regulation, origins
  of life, non-Mendelian kinetics.)

6
A more modern example-protein folding

• Most biological useful proteins are folded into
  compact shapes, on time scales from microseconds
  to seconds.
• Proteins are random heteropolymers of amino acids
  most heteropolymers DO NOT fold into compact
  shapes
• Levinthals paradox (from wikipedia)In 1969
  Cyrus Levinthal noted that, because of the very
  large number of degrees of freedom in an unfolded
  polypeptide chain, the molecule has an
  astronomical number of possible conformations.
  (The estimate 10300 appears in the original
  article). If the protein is to attain its
  corrected folded configuration by sequentially
  sampling all the possible conformations, it would
  require a time longer than the age of universe to
  arrive at its correct native conformation. This
  is true even if conformations are sampled at
  rapid (nanosecond or picosecond) rates.
• Easy estimate from my class 1048 Universe
  lifetimes!!!!!!!!!!!
• Evidently, through evolution, useful proteins
  have been designed to foldwhat are the
  organizing principles?

7
Protein Folding
Tertiary structures with ribbon reps. of a-helix
(red) and b-sheet (green) secondary structures

• Proteins are polymers of the 20 biological amino
  acids whose sequence is encoded in the genome
• After synthesis into the primary sequence they
  commence folding first to have secondary
  structure (commonly a-helices and b-sheets) held
  together by hydrogen bonds between nitrogens and
  oxygens in the backbone
• Biologically useful proteins typically fold
  further into compact structures

8
Folding Funnel

• Proteins are generically glassy- there are
  lots of competing interactions which are
  frustrated
• Minimal frustration leads to a funneled
  landscapethis picture is quantitative and has
  emerged from many model studies

Onuchic and Wolynes
9
Use of concept outside of proteins?Example 1
Zeolites?

• Zeolites flat energy landscapeuseful for
  catalysis, environmental cleanuphow to
  funnel the landscape?
• STRUCTURES
• Framework Chemical Composition -
  SinAlmO2(mn)m-
• m Compensating Cations Na, K, etc.
• Composed of SiO4 and AlO4 tetrahedra connected
  through bridge O atoms
• PROPERTIES
• cavities and pores of molecular dimension
• centers of catalysis

10
Example 2 M-theory?

• There is not an obvious unique solution to the
  M-theory (string theory) vacuum
• From Understanding the Landscape by Michael
  R. Douglas
• In my opinion, the most serious obstacle to
  testing the theory is the problem
• of vacuum multiplicity. This has become acute
  with the recent study of
• the string/M theory landscape. We have a good
  reason to think the theory
• has more than 10122 vacua, the Weinberg-Banks-Abbo
  tt-Brown-Teitelboim-
• Bousso-Polchinski et al solution to the
  cosmological constant problem. Present
• computations give estimates more like 10500
  vacua. We do not even know
• the number of candidate vacua is finite. Even
  granting that it is, the problem
• of searching through all of them is daunting.
  Perhaps a priori selection
• principles or measure factors will help, but
  there is little agreement on what
• these might be. We should furthermore admit that
  the explicit constructions
• of vacua and other arguments supporting this
  picture, while improving, are
• not yet incontrovertible.
• Is the anthropic principle a start at a
  funneled landscape principle here?

11
If the folded state is not the generic protein
state, what is?

• Answer an aggregated state?

12
What we are talking about is novel SELF ASSEMBLED
materials..the BAD
Plaques this is your mind on amyloid
Model cross section
Alzheimers
Parkinsons
Ab42 Fibrils (H. Lashuel) Diameter 10
nm Fibrils have been used to template
nanowires!
Huntingtons
Kuru (prion)
Fatality is correlated with plaques. Plaques are
bundles of fibrils (Pictures Feaney lab,
Harvard)
Fibrils Protein Nanotubes/nanofibers!
(Model H. Saibil see also Perutz)
13
Amyloid diseases 20 known Not
knownmechanism for cell death, toxicity BUT, in
at least two cases, precision biology

• Most proteins involved
• are of unknown function
• Incidence rates for
• Huntingtons and Prion
• diseases are quite
• reproducible,
• Alone among these
• diseases, prion diseases
• can be infectious.

14
Amyloids can be GOOD for some organisms!

• Curli amyloids present in bacteria (E. Coli image
  from Chapman lab, U. Mich.) - can participate in
  stationary phase survival mechanism (Bad-may play
  a role in infection)
• Spider silk manufacture has been argued to be pH
  switched alpha -gt beta amyloid self assembly
• Amyloids appear to be part of some insect eggs
  (silk fibroin)
• Controlled reversible amyloid scaffolds useful in
  tissue engineering (e.g., J. Schneider, U.
  Delaware)

15
So what about amyloidogenic proteins?
Prion protein PrP

• Most have large stretches of random structure or
  are completely random!
• May represent the generic tendency of proteins -
  C. Dobson (vs.well folded monomers selected by
  evolution)
• Stabilization of structure apparently comes with
  aggregation.
• Whether in fibril or oligomer form, there is
  cross-beta structure

Fibril axis ? ? b-sheets
16
PolyQ-Huntingtons and other diseases
Zoghbi Orr, Ann Rev Neurosci 2000
Model with elongation of equilib. nucleus
Extrapolate slow PolyQ lag kinetics to
physiological con- centrations (Chen, Ferrone,
Wetzel, PNAS 2002)
17
Theory as a probe of possible sub-observable
structure I

• All atom MD used to probe stability of left
  handed b-helix for PolyQ (folding to helix not
  possible!) (CHARMM)
• Two layered b-helix not stable within several ns
  of simulation time
• Three layered b-helix is stable out to 10 ns

Number of Qs

• PolyQ diseases have critical insert number of 36
• Aggregation studies of PolyQ suggest critical
  nucleus of 1 (!) monomer (Chen, Ferrone, Wetzel,
  PNAS 2002)
• This is the minimal stable left handed beta
  helical turn (18 residues per turn)
• Is the minimal stable PolyQ a left handed
  b-helix? (But Hear Rappu)

Scherzinger et al, PNAS 1999
(Wanker lab)
Stork et al, Biophysical Journal 2005
18
The Beautiful? Organizing principles for amyloid
matter
19
Organizing Principle 1 Extend minimal
frustration in well ordered proteins by domain
swapping

• Link native contacts on one monomer to
  corresponding native contacts on another
  (champion-D. Eisenberg)
• Example - human cystatin at left (Janowski et
  al., Nat Struct Bio 2001)
• Theory extends minimal frustration concept to
  aggregates - Yang, Cho, Levy, Cheung, Levine,
  Onuchic, Wolynes, PNAS 2004

20
Organizing Principle 2 Steric Zippers (D.
Eisenberg group, Nature 2007)

• Synthesized lots of fragments from amyloid-
  ogenic proteins
• Fibrils from combination of beta sheet stacking
  plus steric zipper (interlocking of well packed
  side chains)

21
Organizing principle 3 Amyloid stucture from
monomeric motifs
PrPSc model
Overlay
1T3D
1T3D stacked in silico

• Appears in multiple bacterial enzymes and insect
  antifreeze proteins (11 on PDB)
• Who ordered that? Unlike a-helix which
  Pauling predicted prior to discovery
• and has local hydrogen bonding (residue j bonds
  to residue j3 or j4) b-helix is
• very nonlocal (residue j bonds to j18)
• b-helix structures easily bond into aggregate
  (edge-to-edge bonding of monomers)

22
Organizing principle 4 Amyloids are
intrinsically slow to form!
23
More precision biology? prion incubation (Slepoy
et al., Phys Rev Lett, 2001 Mobley et al.,
Biophys. J. 2003)
Seed Aggregate Fission
Soft Oligomer/Micelle
Hard/Oligomer
24
Model prions in action
Seed introduced slow initial conversion and
aggregation
Wait a while conversion and aggregation
accelerates
25
More precision biology? prion incubation (Slepoy
et al., Phys Rev Lett, 2001 Mobley et al.,
Biophys. J. 2003)
Seed Aggregate Fission
Fission adds (short) doubling and translates-gt
(BSE best fit)
Distribution of aggregation times
Time to aggregate to critical size N over peak
time
26
From organizing principle to disease - one
possibility - oligomers

• Fibrils are not perfectly correlated with disease
  (many have plaques with no AD, some prion
  diseases have no plaques).
• Fibrils may be protective (collecting aggregate
  away from cells)
• Some oligomers can form pores which permeate
  membrane and let in excess calcium.

27
a
Ab
SOD1
Ab
c
A4V
Arctic (E22G)
?-Synuclein
?-Synuclein
A30P
A53T
28
Consider a spherical mad cow specialize to prions

• Prions alone among all diseases as possibly
  spontaneous (thermodynamically unlucky),
  inherited (fatal familial insomnia), or
  infectious (mad cow, kuru)
• Hypothesized as protein only (Prusiner after
  Griffith, 1967) (UV, proteinase, temperature
  insensitivity)
• Knockout of PrP gene from mice with subsequent
  innoculation of infectious material to brain -gt
  no disease
• Proven from test-tube - can get infection with
  polyanions and PrP (Supattupone group this year
  PNAS)
• Strains-unique phenotypes from same
  sequence-different incubation times, lesion
  distributions - hypothesized to be encoded in
  conformation
• Unlike other amyloid proteins, exponential growth
  of aggregates in vivo
• Minimal infectious unit 3-6 monomers (Silveira,
  Caughey, Nature 2005)

29
Yeast/Fungal prions proof of strain in
conformation

• Serves useful function-non-mendelian heredity
  passed in division (pink, present, red, absent)
• Exponential growth shown in vivo and in vitro
  (Weissman lab)
• Strain IS in conformation (Weissmann lab, UCSF,
  Nat SB, 2002)

30
Structure of normal PrPC (Wuethrich et al, PNAS
97, 8334 (2000) 97, 8340 2000)
Proposed structure of PrPSc in one case (Wille
et al, PNAS 99, 3993 (2002) Govaerts et al,
PNAS 101, 83422004) (12 angstrom resolution)

• 90-95 homology in mammals
• Observed in all vertebrates
• Binds copper in divalent form
• sites in humans, mice, six in
• cattle

Trimer of left-handed beta helices proposed (see
V. Daggett For alternative model )
31
Proposed b-helical trimer model for minimal
infectious prion particle (UCSF, Govaerts et al.
PNAS 2004)
Loop
1THJ
Raw EM image of infectious prion aggregatenote
faceting!
Signal averaged density (difference) mapnote
3 Fold symmetry
Proposed prion trimer has same size as known
bacterial trimer (1THJ)

• What holds the UCSF model together? Known
  bacterial trimers are held together by
  intermonomer Zn bonding (1THJ) or massive
  hydrogen bond networks (1T3D)

32
Combining organizing principles Domain Swapped
Prion Trimer (DSTP) Model (S. Yang, H. Levine,
J.Onuchic, D.L. Cox, FASEB J, 2005)
UCSF Model DSTP Model
Is Strain encoded in monomer number?

• Mutations
• Hinge prolines implicated in genetic prion
  disease GSS (P102L,P105L in Humans, P101L,P104L
  Mice) and CJD (P105T Humans, P104T Mice)
• These are relatively hydrophobic compared to P
  hydrophobic interaction possible for DSTP not for
  BPT

• Carried out All atom MD on Domain Swapped Prion
  Trimer (DSTP) for 1 ns (AMBER8)
• Found increased hydrogen bonding in core relative
  to original beta helical trimer model

33
The plot thickens test tube grown fibrils
(Saibil et al, JMB 06)
For this model, M129 Contacts D178!!!
34
C terminal stability good (AMBER)
35
Templating - possible connection to kinetics

• Roughly, extra H bond to link M129 to H177, N178
  in FFI
• Hard to link R177 for dogs to this
• For mice the suspicion is that the S143N change
  relative to humans leads to a different preferred
  thread

For this model, M129 Contacts D178!!!
36
Slight modulation of C-term thread to accommodate
sugars-hard to get all acidic bases OUT
Di gly Ns out
Di gly
Mono gly
Un gly
37
pH dependent switching of C-terminal beta helix?

• Left Sheep prion
• Heat capacity at pH
• 4.5-5
• Right Sheep prion
• Heat capacity at pH
• 3-4 - new intermediate
• (from Rezaei et al, JMB
• 2002)
• PrP output and intake
• Is in low pH vesicles (not
• 3-4 though)
• With kinetic model of
• pH dependent switching
• Can easily get exponential
• Growth from oligomeric
• Autocatalysis

38
Plenty of open questions, besides disease

• Prions in memory? (Si, Lindquist, Kandel, Cell
  2003) pH dependent switching gives at least
  possibility for some reversibility (aplysia CPEB
  acts like prion when expressed in yeast-plays a
  role in aplysia neuronal connection)
• Nonmendelian heredity in prion conformation is
  inefficient way to confer novel phenotype
• Protein genome in extreme environments?

39
Prions in Cancer?

• P53 protein - regulates cell cycle restart and
  programmed cell death (apoptosis)
• Mutations in p53 associated with 50 of cancers
  including breast, prostate, lung
• Two regions of the protein are susceptible to
  amyloid aggregation!
• Is it possible that the p53 acts like a prion
  (more like in yeast)?
• Can the prion kinetics correspond to initial
  tumor formation kinetics? Question to study with
  Diana Qiu this summer.

40
Amyloids in AIDS?

• Protein in semen can fragment in vivo and form
  amyloid
• Amyloid binds the HIV virus
• In lab animals, infectivity of amyloid HIV
  enhanced over HIV alone by 10-100!
• Possible explanation of enhanced transmission
  from male to female relative to female to male
• What is the structure of the amyloid and nature
  of the binding to HIV? Jonathan Lawton, UCD
  undergrad taking a look.

41
Consider a Spherical Mad Cow Physical modeling
of amyloid diseases

• D.L. Cox, Physics, UC Davis
• R.R.P. Singh, Physics, UC Davis
• K.Kunes, S. Dai, J. Romnes, N.R. Hayre, C.
  Trevisan, Physics, UC Davis
• A. Slepoy, Sandia Labs
• R. Kulkarni, Physics, Virginia Polytechnic
  University
• D. Mobley, Pharmaceutical Chemistry, UCSF
• F. Pazmandi, Sidney Austin LLP (Patent Law,
  Intellectual Property)
• S. Yang, U. Chicago Med School
• S. Clark, Oregon State
• E. Olson, Central College Iowa
• H. Levine, J. Onuchic, Center for Theoretial
  Biological Physics, UCSD
• Support NIH (Seed award from regional
  Alzheimers center), NSF (NEAT IGERT, CTBP,
  ICAM), US Army CDMRC

42
Physical modeling of Prion Diseases
or Consider a Spherical Mad Cow!

• D.L. Cox, Department of Physics, UC Davis
• and Center for Theoretical Biological Physics, UC
  San Diego
• R.V. Kulkarni, Physics, Virginia Polytechnic
  University, D. Mobley, Pharmacology, UC San
  Francisco,J. Pan, Physics, UC Davis, K. Kunes, UC
  Davis, Cynthia Trevisan, UC Davis, Robert Hayre,
  UC Davis, Ryan Wintergate, UC Davis, Sunny Dai,
  UC Davis, Jamie Romnes UC Davis,
• R.R.P. Singh, Physics, UC Davis, A. Slepoy,
  Sandia National Labs
• Scott Clark, Oregon State University (REU Student
  2006), E. Olson, Central College Iowa, REU
  Student 2007
• S. Yang, H. Levine, J. Onuchic, CTBP UC San Diego
  
• Support U.S. Department of Energy
  (Metalloproteins), N.S.F. (IGERT on Nanoparticles
  in the Environment, Agriculture, and Technology,
  CTBP), U.S. Army Congressionally Mandated Medical
  Research Fund, NIH Regional Alzheimers Research
  Institute at UC Davis Medical Center, Guggenheim
  Memorial Foundation

43
Outline

• Overview Protein folding, misfolding, and
  aggregation
• Universality in prions and amyloid diseases
  overview of what is known
• Simple stat mech models and incubation times of
  prion diseases
• Electronic structure calculations on binding
  energies of metal ions and local structure
• New proposed structure for minimal infectious
  unit of prion disease

44
Misfolding of Proteins Amyloid Plaques and
fibrils
Model cross section
Alzheimers
Parkinsons
Ab42 Fibrils (H. Lashuel) Diameter 10
nm Fibrils have been used to template
nanowires!
Huntingtons
Kuru (prion)
Fibrils Protein Nanotubes! (Model H.
Saibil see also Perutz)
Plaques are bundles of fibrils (Pictures
Feaney lab, Harvard)
45
Fibrils vs. oligomers in toxicity
Prion areal oligomer aggregates and fibrils H.
Wille et al, PNAS 99, 3993 2002
Ab42 fibrils via carbon nanotube AFM Leiber
group, Harvard

• Fibrils Common and more easily studied post
  mortem feature (compose plaques)not unique nor
  always seen
• Oligomers Hard to see, not known if on pathway
  to fibrils or off, but emerging as probable
  source of toxicity (more on Ab later) --Protein
  Nanocrystals

46
Amyloid diseases misfolding of proteins
47
So what about amyloid proteins?

• Most have large stretches of random structure or
  are completely random!
• Stabilization of structure apparently comes with
  aggregation.
• Whether in fibril or oligomer form, there is
  cross-beta structure

Fibril axis ? ? b-sheets
48
What is special about prions?

• Prion Proteinaceous infectious particle
  (Prusiner 1980s).
• Along among amyloid diseases infectious as well
  as sporadic, inherited possibilities (PrPSc)
• Numerous experiments (radiation damage,
  UV/temperature/protease/denaturant
  insensitivity.) -gt NO nucleic acids (not a virus
  or bacteria)
• Bolstered by test-tube synthesis of infectious
  protein only prions last year (Baskakov, Prusiner
  et al, Science 2004)
• Prusiner isolated the PrPc protein as key to the
  diseasemice with the gene for PrPc knocked out
  dont get sick on innoculation with infectious
  prion material (PrPSc and PrPC are identical
  after full denaturation-same primary sequence!)
• Examples Scrapies (sheep), Kuru (humans),
  Creutzfeldt-Jakob Disease (CJD) (humans), Mad
  Cow, Chronic Wasting Disease (deer and elk)

49
Structure of normal PrPC (Wuethrich et al, PNAS
97, 8334 (2000) 97, 8340 2000)
Proposed structure of PrPSc in one case (Wille
et al, PNAS 99, 3993 (2002) Govaerts et al,
PNAS 101, 83422004)

• 90-95 homology in mammals
• Observed in all vertebrates
• Binds copper in divalent form
• sites in humans, mice, six in
• cattle

Trimer of left-handed beta helices gives best
model
50
NB Left handed b-helicesa motif crying for a
model!
1T3D
PrPSc model
Overlay
1T3D stacked in silico

• Unlike a-helix which Pauling predicted prior to
  discovery and has local hydrogen
• bonding (residue j bonds to residue j3 or j4)
  b-helix is very nonlocal (residue
• j bonds to j18)
• b-helix structures easily bond into aggregate
  (edge-to-edge bonding of monomers)

51
What did a few physicists find special about the
prion diseases?

• Alone amongst amyloid diseases, prions can be
  spontaneous, heriditary AND infectious
• Prion diseases represent precision biology
  rates of incidence and dose incubation
  distributions are highly reproducible. (1 in
  106 in developed countries get sporadic CJD
  worldwide). Suggests a purely physico-chemical
  model might capture important features of the
  disease
• Simple models can test important questions about
  the disease from this perspective that protein
  conformation (and potentially aggregate
  structure) dictate disease dynamics and properties

52
Some essential issues to explore in modeling

• Autocatalysis vs. Autocatalytic Aggregation
  (cooperative conversion) Strong arguments (Eigen)
  and data legislate against autocatalysis at the
  monomer level conversion upon aggregation is
  more sensible (and supported by our work).
• What aggregate structures and sizes best
  correspond to experiment?
• Fission is critical to explain exponential
  runaway (Masel, 2000). Do aggregate shapes and
  sizes influence this?
• Can infectious and sporadic time scales be
  reconciled in the models?

53
Dependence upon coordination environment
?----------------gt
12 years --------------?1000 years(!) (Kuru?)
(CJD?)

----gt
----gt
qc 3 Seeded
Sporadic
qc1 Seeded Sporadic
qc2 Seeded Sporadic
54
What is the mechanism for exponential growth?

• Membrane must play a role!

55
Role of membrane in toxicity and exponential
growth (fission?)

• Cheseboro et al, Science, 2005 Engineer
  transgenic (Tg) mice with GPI anchor deleted.
• Evidence is that expressed PrPC transport to
  synapse region but are sent off between cells.
• Innoculate mice with a particular lethal dose of
  PrPSc for which wild type (WT) mice get symptoms
  at 150 days.
• Tg mice dont die or get symptoms out to 600
  days, but accumulate infectious prion material in
  between cells! (Later they die from heart failure
  from large aggregates)

WT
56
Exponential growth also requires the membrane!
(Cox, Singh, Yang)

• Short time elongation kinetics without fission or
  autocatalysis gives t2agrees remarkably well
  with Cheseboro et al!
• We estimate PrPCTg 28 X PrPCWT (testable!)

57
What are the surface structures? Proposed
b-helical trimer model for minimal infectious
prion particle (UCSF)
Loop
1THJ
Raw EM image of infectious prion aggregatenote
faceting!
Signal averaged density (difference) mapnote
3 Fold symmetry
Proposed prion trimer has same size as known
bacterial trimer (1THJ)

• What holds the UCSF model together? Known
  bacterial trimers are held together
• by intermonomer Zn bonding (1THJ) or massive
  hydrogen bond networks (1T3D)
• Is there a problem with the loop? Prolines in
  there ought to be straight so there
• is a likely elastic penalty

58
Theory as a probe of possible sub-observable
structure II Domain Swapped Prion Trimer (DSTP)
Model (S. Yang, H. Levine, J.Onuchic, D.L. Cox,
FASEB J, Nov. 2005)
UCSF Model DSTP Model
Is Strain encoded in monomer number?

• Mutations
• Hinge prolines implicated in genetic prion
  disease GSS (P102L,P105L in Humans, P101L,P104L
  Mice) and CJD (P105T Humans, P104T Mice)
• These are relatively hydrophobic compared to P
  hydrophobic interaction possible for DSTP not for
  BPT
• All the mutations also make the loop more
  flexible - can this speed the kinetics?

• Carried out All atom MD on Domain Swapped Prion
  Trimer (DSTP) for 1 ns (AMBER8)
• Found increased hydrogen bonding in core relative
  to original beta helical trimer model

59
The plot thickens test tube grown fibrils
(Saibil et al, JMB 06)
60
What are the fibrils made of?

• Form beta helices from N
• side of prion and C-side of
• prion (peptide always runs
• from N --gt C )
• Excellent stability from C
• side -- constraint of disulfide
• bond for cysteines on corner
• Improved N terminal model
• Good comparisons with beta
• helices from protein data
• bank in AMBER runs
• Scott Clark, REU last year
• suggested N2 idea, developed
• program in Java to examine
• hydrophobicity/hydrofelicity,
• hydrogen bonding, and volume
• packing of model structures--
• co-author on paper being
• submitted to J Molec Biol this

61
How to build the fibrils?
With modest assumptions, only eight ways to build
tetramers -- of these, only IV, III, IV can
fit data
62
Is there more? Does the structure give insight to
the in vivo biology?

• M (methionine) at amino acid 129 is linked to
  disease in humans (where Valine is also possible)
  dominates Kuru cases and human mad cow (vCJD)
• Assume that the remarkably stable C beta helix is
  metastable in vivo--can assist in templating
  the conversion to prion form
• In this fibril model, M129 is at corner of N beta
  helix, and can couple to H177 (histidine) of C
  term beta helix via hydrogen bond
• Fatal Familial insomnia, inherited disease works
  ONLY with M129 and mutation from Aspartic Acid
  (D) to Asparagine (N) at 178 -- in this model,
  extra hydrogen bond --gt potentially faster
  conversion (1000-10000) !
• Dogs Despite the fact that cats, many other
  animals did get mad cow dogs did not -- they
  have what amounts to very long arginine (R ) at
  177 and D at 178 -- very hard to form h-bond
  (1000 times slower)

63
Protein X ?

• If the C-term beta helix is switchable it might
  work like protein X -- the key residues are on
  the top and bottom of the beta helix and render
  it (arguably) stickier for mouse over human
  (testable)
• Possible switch threads with constraint of no
  basic residues pointing (at least on interior
  loops) leads generally to one E pointing in --
  hence could be pH switch between alpha and beta
  conformations.

64
Goal for Evan Olson REU this summer

• Develop stochastic simulation for prion
  conversion model (normal to infectious) involving
  this notion of the switchable C-terminal beta
  helix as ProteinX to template the infectious
  form.

65
Conclusions

• Simple areal aggregation model with little
  biology accounts for much within the protein only
  model!
• Fluctuation dominated incubation time
  distribution
• Difference between infectious and sporadic time
  scales
• Shape (including curvature) of dose-incubation
  distributions
• 1D aggregation less likely (hard to get small
  fission time) but seems to occur between cells.
• Membrane mediates expected exponential growth.
• Domain swapping of the b-helices in the proposed
  prion trimer may stabilize the structure Also
  suggests a model for strains of prions
• Can construct plausible fibril models from
  N-terminal AND C-terminal beta helices
• (1) In turn, these models suggest a connection
  to disease in vivo (templating by the C-terminal
  beta helix)

66
Questions?

• Are prions involved in the molecular basis for
  synaptic plasticity/memory?
• (intriguing suggestions from knockout mice,
  prion like proteins in slugs)
• How on earth does PrP involved in .5 of Cu
  binding shut down neuronal Cu metabolism upon
  infection?

67
Simple model step distribution for single seed
aggregation

• Lag First Arrival to fission size Assume Di
  initial seedstlag is estimated as solution to
• F(Di,tlag) 1 (1 F(1,tlag))Di ½
• which for the step dist. is approximately
• tlag t0 T/2Di
• decreasing with size of initial dose
• Doublings set by time to reach clinical number
  Df
• n2 log2Df log21intDi/(T/t2)
• Net- tinc tlag n2t2 tends to log for
  large dose,
• deviates towards long times for small dose

68
Experimental results for oligomers in Alzheimers

• How can oligomers damage cells?
• Several possible mechanisms
• One possible mechanism

• Membrane insertion
• Formation of oligomeric channels after inserting
• Cell degeneration due to excess Ca2
• Appears to work in vitro

AFM image of A?42 channels in artificial planar
lipid bilayer Lin et al., FASEB 152433
69
Channel structure

• Earlier modeling predicted channels
• A variety possible
• Double-hexamer ?-barrel pores look similar to
  experiment
• Here
• Two helices 15-24, 28-40
• Interlinking from upper and lower leaflets

Image credit Durell et al., Biophys. J.
672137-2145, 1994
70
Questions on experiment

• If this is the mechanism, why doesnt everyone
  get AD?
• Perhaps normally A? doesnt insert easily
  something has to change insertion behavior to
  allow channel formation and cause AD
• FAD mutants cause AD. Do they change insertion
  behavior?
• Only about 7 of AD cases linked to mutations
• But provide tool for assessing toxicity
  mechanisms

71
Background of this model

• Models peptide insertion into lipid bilayers
• Based on a variety of earlier models
• Coarse-grained/mean-field
• Residues are spheres
• Each has polarity, hydrophobicity, and reduction
  in hydrophobicity due to helical folding
• 3.0 Å diameter
• Residues linked by 3.8 Å rigid bonds
• Lipids replaced by potentials
• Monte Carlo, not MD

Tail region Head Water
Maddox and Longo, Biophys. J. 82244
72
Results

• Question FAD mutations cause AD do they change
  insertion behavior?
• Answer Not insertion ease, but possibly inserted
  conformation
• Three basic inserted conformations

73
Results
74
Hypothesis continued

• Would explain why normally, A?40 less toxic than
  A?42
• Testing hypothesis
• Mutations increasing prevalence of conformation
  (c) will be more toxic
• Tested on E22Q,D23N double mutant which (Van
  Nostrand et al.) is more toxic than either alone
• We agree, but within ? of D23N
• Test on E22D A?40 (Melchor et al) which is not
  toxic (HCSM cells) and E22A, which is
• We would predict both would decrease it E22A more

75
Questions?

• Quantitative modeling of strains as encoded by
  aggregate conformation data on yeast prions is
  best right now
• What holds the trimer together, and does domain
  swapping play a role? (S. Yang, H. Levine, J.
  Onuchic)
• Are prions involved in the molecular basis for
  synaptic plasticity/memory?
• How on earth does PrP involved in 1 of Cu
  binding shut down all neuronal Cu metabolism upon
  infection?